The overall objective of this work is to study what controls the phenomenon of X-chromosome inactivation in early female mouse embryos. Our working hypothesis is that methylation of specific sites in X-linked genes is involved in this process. We have carried out a study of the methylation patterns of the mouse Hprt gene when they are carried on the active and inactive X-chromosome. This was accomplished by making use of clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. This characterization revealed two regions of differential methylation in the mouse Hprt gene. First, a 5' region of the gene is completely unmethylated when carried on the active X and is extensively methylated when carried on the inactive X. We have extended this negative correlation between methylation and gene expression by showing that these 5' sites are demethylated when the Hprt gene is reactivated either spontaneously or following 5-azacytidine treatment. Second, we have identified several sites in the 3' 20kb of the gene, extending from exon 3 to exon 9, which are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. Our findings, taken in conjunction with previous studies by others of the human Hprt gene, indicate that there is conservation of the regions of differential methylation between mammalian species and this suggests that these regions might play a role in the regulation of expression and X-chromosome inactivation of the Hprt gene. The goal of our present studies is to determine whether methylation of the sites in the 5' region of the gene occurs concomitant with X-inactivation or if these changes in methylation are perpipheral to the phenomenon, perhaps acting as a final "locking" process. (M)